Inflammation is characteristic of human calcifying diseases, including nephrolithiasis and atherosclerosis. While evidence has linked infectious agents to the inflammation present in atherosclerosis, factors that contribute to the calcification remain to be identified. Preliminary data are presented demonstrating that nanobacteria, a novel, slow-growing microorganism which forms a mineralized shell, can be isolated from diseased, calcified tissues of humans and experimental animals. Therefore, the central hypothesis of this proposal is that nanobacteria are a previously unexpected pathogenic factor in these human diseases. Nanobacteria remain controversial in the scientific community because genetic material has not yet been identified. Therefore, the major goal of this two-year R-21 proposal is to clone and characterize unique nanobacterial DNA sequences in order to unequivocally identify and classify this organism, and to develop definitive genetic tests for evaluating human and experimental tissues. A multidisciplinary team has been created with the long-term goal to isolate nanobacteria from tissue of humans and experimental animals, grow the isolates in culture, and then inoculate experimental animals and cells with the organism to reproduce a calcific response, as required to fulfill Koch's postulates. Distinct advantages of this grant application include the vast experience in Laboratory Medicine for extracting, concentrating, and purifying nucleic acid and developing rapid molecular diagnostic tests for infectious disease agents, as well as the use of cryogenic microcomputer tomography (CT) which allows 3D imaging, histomorphometric/immunological imaging, and genetic probing of a single sample. This is a HIGH-RISK proposal, but if nanobacteria are routinely identified in diseased and calcified renal and atherosclerotic tissue, our understanding of these common human diseases would be revolutionized and HIGH-IMPACT consequences would likely ensue, including new treatment approaches for these and possibly other calcifying diseases.